Abstract
Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson's disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread.
| Original language | English (US) |
|---|---|
| Article number | 109189 |
| Journal | Cell Reports |
| Volume | 35 |
| Issue number | 10 |
| DOIs | |
| State | Published - Jun 8 2021 |
Keywords
- Braak hypothesis
- GWAS
- ITGA8
- high-throughput screen
- siRNA
- weighted gene co-expression network analysis
- weighted protein-protein network interaction analysis
- α-synuclein
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
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In: Cell Reports, Vol. 35, No. 10, 109189, 08.06.2021.
Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein
AU - Kara, Eleanna
AU - Crimi, Alessandro
AU - Wiedmer, Anne
AU - Emmenegger, Marc
AU - Manzoni, Claudia
AU - Bandres-Ciga, Sara
AU - D'Sa, Karishma
AU - Reynolds, Regina H.
AU - Botía, Juan A.
AU - Losa, Marco
AU - Lysenko, Veronika
AU - Carta, Manfredi
AU - Heinzer, Daniel
AU - Avar, Merve
AU - Chincisan, Andra
AU - Blauwendraat, Cornelis
AU - García-Ruiz, Sonia
AU - Pease, Daniel
AU - Mottier, Lorene
AU - Carrella, Alessandra
AU - Beck-Schneider, Dezirae
AU - Magalhães, Andreia D.
AU - Aemisegger, Caroline
AU - Theocharides, Alexandre P.A.
AU - Fan, Zhanyun
AU - Marks, Jordan D.
AU - Hopp, Sarah C.
AU - Abramov, Andrey Y.
AU - Lewis, Patrick A.
AU - Ryten, Mina
AU - Hardy, John
AU - Hyman, Bradley T.
AU - Aguzzi, Adriano
N1 - Funding Information: Imaging was performed with support of the Center for Microscopy and Image Analysis, University of Zurich. Flow cytometry was performed with equipment of the flow cytometry facility, University of Zurich. RNA sequencing experiments and basic data analyses were performed at the functional genomics center Zurich (FGCZ). Flow cytometry was performed at the UCL Joint Great Ormond Street Institute of Child Health and Institute of Ophthalmology Flow Cytometry Core Facility, supported by the Great Ormond Street Children’s Charity (GOSHCC), grant reference U09822 (October 2007), UCL Capital Equipment Fund , School of Life and Medical Sciences (September 2012), and UK Research and Innovation , grant reference MR/L012758/1 (March 2014). FLIM imaging was performed at the UCL Confocal Imaging Facility, division of biosciences, which is funded by the UCL Capital Equipment Fund . E.K. is the recipient of an HFSP long-term fellowship ( LT001044/2017 ), a Dr. Wilhelm Hurka Foundation project grant, and an EMBO , long-term fellowship ( ATLF-815-2014 , which is co-funded by the Marie Curie Actions of the European Commission [ LTFCOFUND2013 and G Α -2013-609409 ]). A.A. is the recipient of an Advanced Grant of the European Research Council (ERC 670958 ) and is supported by grants from the Swiss National Science Foundation (SNF 166943 and 179040 ) plus a Sinergia grant ( 183563 ), and a Distinguished Investigator Award of the NOMIS Foundation . J.H. is supported by the Medical Research Council (award number MR/N026004/1 ), the Wellcome Trust (award number 202903/Z/16/Z ), the Dolby Family Fund , the National Institute for Health Research University College London Hospitals Biomedical Research Centre, and the BRCNIHR Biomedical Research Centre at University College London Hospitals NHS Foundation Trust and University College London. M.R. was supported through the award of a UK Medical Research Council Tenure Track Clinician Scientist Fellowship ( MR/N008324/1 ). J.A.B. is supported through the Science and Technology Agency , Séneca Foundation , and CARM, Spain (research project 00007/COVI/20 ). R.H.R. was supported through the award of a Leonard Wolfson Doctoral Training Fellowship in Neurodegeneration . A.P.A.T. is supported by the Professor Dr. Max Cloëtta Foundation . C.M. and P.A.L. are supported by the Biomarkers Across Neurodegenerative Diseases Grant Program 2019, BAND3 ( Michael J. Fox Foundation , Alzheimer's Association , Alzheimer’s Research UK , and Weston Brain Institute [grant number 18063 ]). This work was supported by the UK Dementia Research Institute , which receives its funding from DRI Ltd , funded by the UK Medical Research Council , Alzheimer's Society , and Alzheimer’s Research UK . This work was supported in part by the Intramural Research Programs of the National Institute of Neurological Disorders and Stroke (NINDS), the National Institute on Aging (NIA), and the National Institute of Environmental Health Sciences both part of the National Institutes of Health, Department of Health and Human Services; project numbers 1ZI Α -NS003154 , Z01-AG000949-02 , and Z01-ES101986 . The funders played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to thank Gilles Kratzer (UZH Institute of mathematics) for expert advice on statistical analyses, Dr. Kelvin Luk (University of Pennsylvania) for providing us with vials of their HEK QBI WT synuclein cell line, Dr. Berend Snijder for allowing us access to his Opera Phenix for imaging a subset of plates, Dr. Giancarlo Russo for assistance with RNA sequencing analysis and data management, Dr. Christopher Thrasivoulou and Dr. Alan Greig for assistance with FLIM optimization, and Rita Moos and Jacqueline Wiedler for assistance with ordering consumables and grant administration. Funding Information: Imaging was performed with support of the Center for Microscopy and Image Analysis, University of Zurich. Flow cytometry was performed with equipment of the flow cytometry facility, University of Zurich. RNA sequencing experiments and basic data analyses were performed at the functional genomics center Zurich (FGCZ). Flow cytometry was performed at the UCL Joint Great Ormond Street Institute of Child Health and Institute of Ophthalmology Flow Cytometry Core Facility, supported by the Great Ormond Street Children's Charity (GOSHCC), grant reference U09822 (October 2007), UCL Capital Equipment Fund, School of Life and Medical Sciences (September 2012), and UK Research and Innovation, grant reference MR/L012758/1 (March 2014). FLIM imaging was performed at the UCL Confocal Imaging Facility, division of biosciences, which is funded by the UCL Capital Equipment Fund. E.K. is the recipient of an HFSP long-term fellowship (LT001044/2017), a Dr. Wilhelm Hurka Foundation project grant, and an EMBO, long-term fellowship (ATLF-815-2014, which is co-funded by the Marie Curie Actions of the European Commission [LTFCOFUND2013 and G?-2013-609409]). A.A. is the recipient of an Advanced Grant of the European Research Council (ERC 670958) and is supported by grants from the Swiss National Science Foundation (SNF 166943 and 179040) plus a Sinergia grant (183563), and a Distinguished Investigator Award of the NOMIS Foundation. J.H. is supported by the Medical Research Council (award number MR/N026004/1), the Wellcome Trust (award number 202903/Z/16/Z), the Dolby Family Fund, the National Institute for Health Research University College London Hospitals Biomedical Research Centre, and the BRCNIHR Biomedical Research Centre at University College London Hospitals NHS Foundation Trust and University College London. M.R. was supported through the award of a UK Medical Research Council Tenure Track Clinician Scientist Fellowship (MR/N008324/1). J.A.B. is supported through the Science and Technology Agency, S?neca Foundation, and CARM, Spain (research project 00007/COVI/20). R.H.R. was supported through the award of a Leonard Wolfson Doctoral Training Fellowship in Neurodegeneration. A.P.A.T. is supported by the Professor Dr. Max Clo?tta Foundation. C.M. and P.A.L. are supported by the Biomarkers Across Neurodegenerative Diseases Grant Program 2019, BAND3 (Michael J. Fox Foundation, Alzheimer's Association, Alzheimer's Research UK, and Weston Brain Institute [grant number 18063]). This work was supported by the UK Dementia Research Institute, which receives its funding from DRI Ltd, funded by the UK Medical Research Council, Alzheimer's Society, and Alzheimer's Research UK. This work was supported in part by the Intramural Research Programs of the National Institute of Neurological Disorders and Stroke (NINDS), the National Institute on Aging (NIA), and the National Institute of Environmental Health Sciences both part of the National Institutes of Health, Department of Health and Human Services; project numbers 1ZI?-NS003154, Z01-AG000949-02, and Z01-ES101986. The funders played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to thank Gilles Kratzer (UZH Institute of mathematics) for expert advice on statistical analyses, Dr. Kelvin Luk (University of Pennsylvania) for providing us with vials of their HEK QBI WT synuclein cell line, Dr. Berend Snijder for allowing us access to his Opera Phenix for imaging a subset of plates, Dr. Giancarlo Russo for assistance with RNA sequencing analysis and data management, Dr. Christopher Thrasivoulou and Dr. Alan Greig for assistance with FLIM optimization, and Rita Moos and Jacqueline Wiedler for assistance with ordering consumables and grant administration. Acquired funding, A.A. E.K. B.T.H. J.H. M.R. and P.A.L.; supervised study, A.A. B.T.H. J.H. M.R. P.A.L. J.A.B. E.K. and A.P.A.T.; performed clonings, E.K. Z.F. J.D.M. and A.W.; performed and optimized HTS imaging, E.K. and C.A.; optimized and performed wet-laboratory experiments for HTS, E.K.; performed and optimized confocal imaging, E.K.; printed siRNAs, E.K. M.C. M.E. M.A. D.H. A. Carrella, D.B.-S. L.M. D.P. and A.W.; analyzed GWAS data, S.B.-C. and C.B.; analyzed gene expression data, J.A.B. E.K. K.D. R.H.R. M.R. and S.G.-R.; analyzed protein-protein network interaction data, C.M. and P.A.L.; performed flow cytometry, E.K. and A.W.; performed FACS, M.L. V.L. E.K. and A.W.; performed western blot, E.K.; performed qPCR, E.K.; performed tissue culture, E.K. A.W. S.C.H. and J.D.M.; performed FRET and FLIM experiments, E.K.; performed mitochondrial experiments, A.Y.A. and E.K.; optimized and maintained robotics and provided critical advice on robotics usage, M.E.; analyzed wet-laboratory data, E.K. A. Crimi, A.W. and A. Chincisan; wrote code for analysis of HTS data, A. Crimi and A. Chincisan; RNA sequencing, A.D.M. D.H. M.A. and E.K.; wrote the manuscript, E.K. and A.A.; edited manuscript, all authors. B.T.H. has a family member who works at Novartis, and owns stock in Novartis; he serves on the scientific advisory board (SAB) of Dewpoint and owns stock; he serves on an SAB or is a consultant for Avrobio, AZTherapies, Biogen, Novartis, Cell Signaling, the U.S. Department of Justice, Takeda, Vigil, W20 Group, and Seer; and his laboratory is supported by sponsored research agreements with Abbvie and F-Prime and has research grants from the National Institutes of Health, Cure Alzheimer's Fund, Tau Consortium, and the JPB Foundation. Publisher Copyright: © 2021 The Authors
PY - 2021/6/8
Y1 - 2021/6/8
N2 - Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson's disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread.
AB - Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson's disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread.
KW - Braak hypothesis
KW - GWAS
KW - ITGA8
KW - high-throughput screen
KW - siRNA
KW - weighted gene co-expression network analysis
KW - weighted protein-protein network interaction analysis
KW - α-synuclein
UR - http://www.scopus.com/inward/record.url?scp=85107937682&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85107937682&partnerID=8YFLogxK
U2 - 10.1016/j.celrep.2021.109189
DO - 10.1016/j.celrep.2021.109189
M3 - Article
C2 - 34107263
AN - SCOPUS:85107937682
SN - 2211-1247
VL - 35
JO - Cell Reports
JF - Cell Reports
IS - 10
M1 - 109189
ER -